Methods for rapid microbial detection

ABSTRACT

A method of testing for target bacteria involves adding bacteriophage to a sample to infect the bacteria in the sample; killing extracellular bacteriophage without at the same time killing phage-infected bacteria; amplifying bacteriophage remaining in the sample; and causing the bacteriophage to infect reporter bacteria and thereby produce an observable signal. The reporter bacteria are genetically engineered to have an indicator gene which on expression gives rise to a detectable signal, wherein expression of the indicator gene is initiated on bacteriophage infection of the bacteria.

FIELD OF THE INVENTION

This invention concerns methods for the detection of microbialorganisms, e.g. bacteria and bacteriophage in a wide variety of casessuch as foods, clinical specimens and environmentally important samples.The invention can also be applied to examine susceptibility toantibacterial compounds and the effectiveness of virucidal agents.

BACKGROUND OF THE INVENTION

The detection and identification of bacteria is of great interest in avariety of microbiological applications. For example the need to screenfood, water and other beverages for pathogenic bacteria is crucial inensuring consumer safety. The determination of levels of certainfamilies of bacteria is a commonly used approach to estimating the shelflife and microbial acceptability of such products and hygienic status ofthe processing equipment and raw materials used in their manufacture.The diagnosis of microbial infections also relies on the detection ofthe causative organism. The screening of environmental waters fororganisms such as Legionella has recently assumed considerableimportance.

The desire to detect bacteriophages (viruses which specifically infectbacteria) stems from their ability to kill bacteria and hence thedeleterious effect they can have on the fermentation of milk, forexample, by killing the starter culture bacteria. Bacteriophages arealso used, e.g. in the water industry, as tracers to determine the rateof river flow or sewage leakage.

The methods available to carry out bacterial detection and enumerationsuffer from a number of drawbacks. Traditional culture based methodsform the backbone of the tests used but as they rely on bacterialgrowth, often in selective media that allows the desired organism togrow while suppressing the growth of other bacteria, they are inherentlyslow; a total viable count taking 18-24 hours and detection ofSalmonella taking 4-7 days. In many cases bacterial numbers may beunder-estimated because their particular growth requirements may not bemet by the media provided or they may have been sub-lethally injured orentered a stress induced physiological state in which they are viablebut not culturable. Culture based methods are not suitable for on-sitetesting due to the long incubations required.

A variety of methods have been proposed to address these drawbacks andallow rapid bacterial detection, some claimed to be applicable toon-site use. For example, the measurement of adenosine triphosphate(ATP), an intracellular component of all living things, provides a rapidmethodology but this is not specific and hence offers, at best, anestimation of the total bacterial population.

Immunoassay approaches with antibodies specific for the desired bacteriahave failed to achieve widespread use because of inadequate specificityand sensitivity leading to the need for two days of enrichment culturebefore the immunoassay in the case of a Salmonella test, for example.Interference from competing organisms and the sample matrix have led tounacceptable rates of false positive and false negative results andprotocols that are not substantially shorter than culture.

Methods based on DNA or RNA probes have been applied to bacterialdetection but currently suffer from the problem of involving complicatedprotocols, unpleasant chemicals in some of the solutions and the needfor elevated temperature. They certainly are not user friendly totechnicians trained in classical microbiology. Together withimmunoassays and nucleic acid amplification strategies such as thePolymerase Chain Reaction (PCR) they do not distinguish between live anddead bacteria. This makes them unsuitable for direct assays (where thereis no culture step to allow the amplification of the living organisms)of the viable bacteria. In certain applications this distinction is veryimportant e.g. when disinfectants have been used to ensure that thereare very few living Legionella in a water system, it is meaningless touse an assay which fails to discriminate and detects the organisms whichhave been killed by the disinfection process.

There are a number of approaches that rely on expensive instruments tospeed up the detection of bacteria. An example of these isimpedance/conductance measurement where the bacterial presence isdetected by their metabolism of complex nutrients to simpler chemicalswith a concomitant change in the electrical properties of the medium.Such methods are highly capital intensive and inappropriate for smalllaboratories or on-site use.

Microscopy techniques, possibly employing selective staining, arelimited in sensitivity and generally offer poor differentiation betweenliving and dead bacteria. Routine microscopy will only permitpresumptive identification based on morphology unless combined withselective culture or immunological staining.

In view of the above, it is highly desirable to have methods for thedetection of bacteria and bacteriophages that are simple to perform,specific, rapid (providing results in hours rather than days), able todetect only living organisms, capable of on-site use and without theneed for an expensive instrument. Preferably the assays would beperformed on a wide variety of sample types without pre-treatment andwith a minimum number of steps. The assay result should be a detectableevent that is easily observed and amenable to automated reading. Itwould be further desirable if the assays were able to detect disabledbacteria which might otherwise require a pre-enrichment culture step anda selective enrichment step for detection. Non-culturable but viableorganisms should also be detected.

DESCRIPTION OF PRIOR ART

Many of the methods to identify microbial organisms have been based onclassical microbiology using nutrient agar plates. In recent yearsattempts to use molecular biology and genetic modification have alsobeen applied to this area. In particular Ulitzur and Kuhn (Europeanpatent application 0168933) introduce a detectable marker, often theenzyme luciferase, into bacteriophages which can then be used forbacterial detection. Modified phages are added to samples suspected ofcontaining the bacteria of concern which is a host for thatbacteriophage. If a suitable bacterial host is present thenbacteriophage nucleic acid will infect that host and will be expressedin the bacteria. When the modified bacteriophage carries the markerluciferase then the presence of the bacteria can be determined by theemission of light which can be easily measured.

PCT/90/04041 by DNA Plant Corporation uses a different marker systemi.e. ice nucleation and also a panel of phages to allow typing.

PCT90/04037 uses genetically modified microorganisms as indicators in atest system for the detection of a range of toxic substances.

PCT89/03878 describes a system based on genetic modification which canbe applied to eukaryotic viruses.

U.S. Pat. No. 4,797,363 uses bacteriophages which have been labelledwith a variety of signal systems. However, the labelling is by directchemical methods and does not depend on expression of additional genes.

ADVANTAGES OF THE NEW INVENTION

In systems based on genetic modification using bacteriophage to detectbacteria, the genetic modification has been to the bacteriophage. Forvarious technical reasons this is not always an easy modification tomake. If a panel of phages is required for bacterial identification theneach phage of the panel will need the similar modification and so theproblem is multiplied. One embodiment of this invention allows use ofnon-genetically modified bacteriophages with the later use of a modified(or panel of modified) bacteria carrying the detectable marker. It istechnically far easier to modify the bacteria compared with thebacteriophage.

SUMMARY OF THE INVENTION

The invention comprises several methods for the testing and thedetection of bacteria or bacteriophages in a sample. It also allows thesusceptibility of bacteria to antibacterial agents to be determined andthe effectiveness of virucidal agents to be assessed. Both qualitativeand quantitative testing are encompassed.

This is achieved by exploiting the interaction between bacteriophagesand bacteria. The way in which a bacteriophage infects a bacterium canbe used to develop assays. The interaction is specific and once thisrecognition/binding event has taken place, the bacteriophage injects itsnucleic acid into the host bacterium. The host is then used to replicatethe `phage` being produced and, upon breaking open the host, to theninfect additional bacteria.

Once the phage has specifically infected the cell and injected itsnucleic acid, it is protected from the extracellular environment. Partof the invention makes use of this to kill or remove those phages whichhave not specifically infected a bacterium. Therefore if the samplecontains, say, Salmonella then the specific phage will be protected, ifSalmonella is absent no phage will be protected. The removal or killingof unbound phage can be achieved by a variety of methods for examplevirucidal agents, heat, removal of chemicals essential for phagestability etc.

This aspect is clearly very different from other diagnostic approachesusing phages where a directly or indirectly labelled phage is allowed tobind to the surface of a bacterium and unbound is washed away prior todeveloping a signal (enzymatic, fluorescent, luminescent, etc.). In thiscase all the important events occur outside the cell contrasting clearlywith the present invention.

The next stage of the invention depends on the number of cells beingdetected. The number of bacteriophages protected and able to replicateand emerge may be sufficient to be detected directly. If not the numbercan be amplified by growing them on a propagating host for the requiredtime (this can be short since phage generation times are less than 1hour and 10-1000 progeny are produced).

Once the number of phages is adequate for detection, this can beachieved by a number of methods e.g. immunologically with an antibody tosome component of the phage or with a nucleic acid probe to the phagegenome or by plaque assay. A preferred method is based on the discoverythat a bacterium can be constructed by genetic modification that has thepotential to produce a detectable signal (gene(s) coding for a phenotypethat can be readily detected) but this potential is only expressed whenthe bacterium is infected by a phage. The phage triggers the signalgeneration and hence the presence of the phage (and therefore of thebacterium that protected it) can be detected sensitively and easily.These bacteria, herein called reporter bacteria, are new materials perse and constitute another aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a map of plasmid pSB292 containing the pCK1 gram positivereplicon and a promotorless copy of the luxAB genes.

FIG. 2 depicts the bioluminescence profile of pSP19 following phageinfection vs. control without infection.

DETAILED DESCRIPTION

The invention thus includes several methods of testing which involve theuse of one or both of two key features. One key feature is the killingof extracellular bacteriophage in a fluid sample which also containsphage-infected bacteria. The other key feature is the provision ofreporter bacteria which are genetically engineered to have an indicatorgene which on expression gives rise to a detectable polypeptide,Expression of the indicator gene is initiated only on bacteriophageinfection of the bacteria. The nature of the indicator gene, and themeans used to detect the detectable polypeptide, are not material to theinvention.

There follows sections describing these two key features. These are thenfollowed by a description of various methods of testing according to theinvention.

Differential phage killing

Hirotani et al. (1991) described the anti-viral activity of unsaturatedfatty acids and related alcohols against T5 phage. With the addition of3000 lux (Lumens per square meter) illumination, they achieved 97.6%inactivation of T5 phage with C18:2 linoelaidic acid (LA) at 50 μg/ml.We have demonstrated that a protocol along these lines achieves thedifferential phage killing that we desire. The fluid sample containingthis acid or alcohol addition is subjected to photon irradiation,fluorescent light being perfectly suitable. If monochromatic light isused, the wavelength is preferably around 420 nm, although a subsidiaryeffect is seen at around 530 nm. Illumination of sufficient time andintensity is effective to selectively kill extracellular bacteriophage,without adversely affecting phage which have infected bacteria in thesample.

An alternative treatment for differential phage killing involvesaddition to the sample of a C1 to C4 carboxylic acid such as aceticacid. The acetic acid concentration in the sample is preferably from0.01 to 1.0%, particularly 0.1 to 0.5%, the figures being expressed aspercent by volume of glacial acetic acid in the sample. If theconcentration is too low, extracellular bacteriophage are not killed; ifthe concentration is too high, infected bacteria in the sample may bedamaged. The sample is incubated for a time to permit the acetic acid toeffect virucidal action, e.g.37° C. for 15 minutes, and is then broughtback to approximate neutrality by the addition of base. Vinegar (5%) hasbeen used in place of acetic acid with very Similar results.

After killing extracellular bacteriophage, it is often necessary toamplify surviving phage (present within infected bacteria) in thesample. However, it appears that bacteriophage infection of bacteria atambient or higher temperatures may sensitise the bacteria to subsequentexposure to acetic acid, so that the bacteria subsequently fail toamplify the phage properly. A preferred solution to this problem is toeffect bacteriophage infection of bacteria in the sample at atemperature below ambient. For example, the sample can be held at 0° C.in an ice-bath. Phage infection takes place efficiently even at theselow temperatures, and the resulting infected bacteria are no longersensitive to the acetic acid treatment.

For those organisms which are themselves acid sensitive, e.g.pseudomadaceae, such treatment may not be effective. A possiblealternative treatment involves use of a mixture of hydrogen peroxide andsodium hydroxide.

Preparation of Reporter Bacteria

Successful bacteriophage (phage) infection generally requires thesequential expression of sets of genes. Studies of a number ofbacteriophages have identified a variety of biological strategies forobtaining temporal regulation (e.g. see Rabussay and Geiduschek, 1977;McKnight and Tjian, 1986). Most of these strategies involvetranscriptional control. The classically described strategies operate atinitiation of transcription (Jacob and Monod, 1961; Ptashne, 1986).

In an alternative mechanism, first described for phage Lambda, the samepromoters used for transcription of genes expressed early in theinfection are used to transcribe genes expressed later through a processof antitermination of transcripts (Roberts, 1969). Operons exhibitingthis type of control are arranged so that genes locatedpromoter-proximal to the termination signal can be expressed maximallywhile genes promoter-distal to the termination signal are unexpressed orpoorly expressed. These latter sets of genes are expressed when somephysiological or developmental change eliminates the activity of thetermination signal. The nature of the termination signals and the natureof the antitermination factors vary greatly (Platt, 1986).

Using the tools and techniques of molecular microbiology, it is possibleto dissect a bacteriophage genome and to clone elements of that genomeinto autonomously replicating plasmid vectors. Among a library of suchrandomly generated constructs, those containing a bacteriophage promotercould express promoter proximal genes; always providing that thepromoter utilised the normal rifampicin sensitive bacterial RNApolymerase. If the cloned bacteriophage DNA contained a terminationsignal in addition to a promoter, genes proximal to the promoter wouldbe expressed but genes distal to the promoter terminator would not.These promoter distal genes could only be expressed if the correctbacteriophage encoded antitermination signal were provided and this maywell be absent on the cloned segment of DNA. The transactingantitermination factor could be provided, however, as a natural part ofthe temporally controlled gene expression commensurate withbacteriophage infection. In other words plasmid-cloned, promoter-distalgenes that are downstream of a transcription terminator could beexpressed only during bacteriophage infection. If the naturallyoccurring bacteriophage genes are replaced by an indicator gene, such asluxAluxB, whose product is easily monitored, then phage dependentantitermination will result in expression of the indicator genephenotype. For a construct employing luxAluxB this would mean that theinitial plasmid vector construct would be dark but that duringbacteriophage infection bioluminescence would result. Although luxAluxBexpression and bioluminescence is a preferred indicator system, otherindicator systems that facilitate monitoring a trans-actingantitermination event following bacteriophage infection could beemployed.

Existing knowledge of the molecular architecture and gene controlmechanisms of bacteriophages such as Lambda O80 , P2, P22, Hk022 and 21would allow those skilled in the art to identify specific DNA regions tobe isolated in vitro for engineering a construct as defined above. This`in depth` knowledge, although helpful, is not necessary, however, asrandom cloning of the genome of a genetically uncharacterisedbacteriophage can provide equally effective constructs.

The bacteriophage 23074-B1 (ATCC) is a Listeria monocytogenesserotype 4phage for which the genome has not been characterised. A partial digestusing the restriction enzyme Sau3a was performed on the genome of23074-B1 and the DNA fragments ligated into the BamH1 site of plasmidpSB292, containing the pCK1 Gram positive replicon (Gasson & Anderson,1985) and a promoterless copy of the luxAluxB genes (FIG. 1). Theligation reaction was used to transform Listeria monocytogenes 23074 tochloramphenicol resistance and recombinant clones were screened for abioluminescent phenotype. Those clones having no constitutivebioluminescence (dark clones) were further screened for bioluminescencefollowing infection with bacteriophage 23074-B1. Clones providing phagedependent bioluminescence were identified. FIG. 2 shows thebioluminescence profile of one such construct (pSP19) following phageinfection. A 1000-fold difference in bioluminescence was observedbetween phage infected and non-infected cells. pSP19 and constructs withsimilar phenotypes provide the basis for detecting the presence ofbacteriophage 23074B1. pSP19 was constructed with out prior knowledge ofthe phage genome and demonstrates the generic nature of the selectionprocess. Other bacteriophages for Listeria monocytogenes could bedetected using a similar protocol.

It is known that not all bacteriophages control temporal gene expressionby antitermination. T7 , for example, provides a new rifampicinresistant RNA polymerase that promotes transcription from novel T7 RNApolymerase-specific bacteriophage promoters (Chamberlin et al., 1970).Other phages of the T7 type would, if screened by a cloning programme asdescribed above, nevertheless provide recombinant constructs silent forexpression of the indicator gene unless infected by the correspondingbacteriophage. Such constructs would have the bacteriophage-specific RNApolymerase promoter sequence proximal to the indicator gene. The hostRNA polymerase would not transcribe such a construct and hence, if theindicator used was luxAluxB, the recombinant would be dark.Bacteriophage infection would, however, lead to the temporal expressionof the bacteriophage RNA polymerase and hence active transcription ofthe indicator gene. The use of T7, T3, T5 and SP6 phage specific RNApolymerases to control gene expression on recombinant plasmid vectors iswell known (Old and Primrose, 1989). It has not, however, previouslybeen used to enumerate bacteriophage and this element is but onepossible control circuit in a generic approach to harnessing temporalexpression to induce, in trans, the expression of an indicator gene.

Bacteriophage P1 is a well characterised phage that appears to useneither termination nor a specific RNA polymerase to temporally regulatephage gene expression (Yarmolinsky and Sternberg, 1988). Nevertheless,promoters have been identified from P1 that are inactive in uninduced P1lysogens but become active about 30 min after prophage induction (ibid).It is possible to predict that these promoters would be identified in ascreening procedure such as that described above for bacteriophage23074B1. The cloning vector would have to be functional in a P1 hostbacterium, but the principle of selection would be the same.

From those bacteriophage currently well characterised at the geneticlevel, there are none that are seen to lack elements of temporalcontrol. It is reasonable, therefore, to postulate that such elementsexist in all bacteriophages. Although mechanisms of temporal controldiffer, all currently identified mechanisms are amenable to theconstruction of bacteriophage/indicator chimeras that place indicatorgenes such as luxAluxB under the expression control of a temporallyregulated bacteriophage gene switch. When present as a stable geneticconstruct in a non-phage infected host, the indicator gene will not beexpressed. During phage infection, however, the bacteriophage can supplya trans-acting factor that activates the expression of the chimericindicator construct. Measurement of indicator expression, for example invivo bioluminescence if luxAluxB is employed, is a direct andquantitative measure of the presence of virulent bacteriophageparticles. Although a detailed understanding of the molecular biology ofa bacteriophage would assist in the construction of the chimericindicator, the demonstration of such a construct for the L.monocytogenesphage 23074B1 shows that a chimeric indicator can begenerated from an entirely uncharacterised bacteriophage. In principle,therefore, chimeric indicator constructs could be generated for anybacterium/bacteriophage couple.

These techniques give rise to reporter bacteria, namely geneticallyengineered bacteria constructed to have a gene switch/indicator genechimera where activation of the switch is dependent upon a trans-actingfactor supplied during phage infection. These genetically engineeredbacteria may be rendered non-viable and non-culturable but retain theircapacity to detect phage infection.

Various methods of testing involving use of these reporter bacteria aredescribed in the following section.

1. The rapid detection of bacteria

a) Phage amplification and assay

Bacteria such as Salmonella spp. or Listeria spp. require recovery andenrichment before detection. Enrichment to bring bacterial numbers up toa detectable level requires a time that is dependent on the rate ofmicrobial growth and division. Increases in bacterial numbers follow anexponential curve and hence for 1 Salmonella or Listeria to reach 10⁸bacteria respectively would take 27 divisions and, with a growth rate of30 minutes, this would require a minimum 14 hours. By comparison, abacteriophage infection cycle typically takes 40 minutes and producesbetween 10 to 100 progeny phage. A single Salmonella or Listeriainfected with a lytic phage could, in the presence of additional helperbacteria, produce 10⁸ bacteriophage particles in 5.3 hours at a burstsize of 10 or in 2.6 hours at a burst rate of 100. In other words,bacteriophage are amplified between 3 and 5 times as fast as bacteria.This amplification of bacteriophage could be used to detect the presenceof low numbers of pathogenic bacteria in foods and other samples asfollows:

25 g of food containing 1-10 pathogenic bacteria would be homogenised ingrowth medium and a pathogen-associated bacteriophage added at aconcentration sufficient to ensure rapid infection of the pathogen(m.o.i.* of 10 or greater) (m.o.i.*: multiplicity of infection). After10 to 15 mins to allow injection of phage DNA into the pathogen, achemical or physical treatment would be used to destroy, remove,neutralise or inactivate all remaining bacteriophage. Examples ofchemical treatment could include virucidal but sub-antimicrobial levelsof biocides. Examples of physical treatment include virucidal butsub-antimicrobial levels of heat. After destruction of bacteriophage,chemical virucides would be neutralised and, if heat was utilised,temperatures returned to those optimum for microbial growth. The initialpathogens would now be phage infected and there would be no viableextracellular phage present. After a further 30 to 40 minutes ofincubation the phage infection cycle in the initial pathogens would becomplete and virulent bacteriophage would be released as the pathogenslyse. The numbers of bacteriophage released would be too small todetect, but these few phage could be amplified if a permissive host (notnecessarily pathogenic) was added to the culture. The addition of 10⁶-10⁷ permissive bacteria would allow amplification of 10- 100 phageparticles or greater in 3 to 5 hours. The presence of bacteriophage atsuch levels could be rapidly and conveniently detected by thebacteriophage assay format described above.

If food or other samples contain low levels of target pathogens, thesepathogens will be indirectly detected by a positive bacteriophage assayfollowing infection and amplification. The preferred assay forbacteriophage would be by via induction of a bioluminescent phenotype ina bacterium genetically engineered to contain a promoter/luxAB orluxAluxB chimera, dependent for expression on phage infection.Immunological assays for the presence of phage in the final amplifiedculture medium could also be considered however.

Samples that contain no target bacteria cannot protect (eclipse) anybacteriophage from the virucidal treatment. No phage would be availabletherefore to be amplified by the permissive bacterial host. Such sampleswould contain no bacteriophage in the final amplified culture andsamples from such a culture would be negative in the bacteriophage assaydescribed above.

Samples could be scored as positive or negative for a target pathogenwithin 3 to 6 hours on the basis of presence or absence of pathogenassociated bacteriophages.

Since bacteriophages may infect and replicate in viable butnon-culturable bacteria, the rapid detection of bacteria should includebacteria sublethally injured. Avoidance of any need to recover growthpotential in sub-lethally injured bacteria, contributes significantly tothe speed of the assay.

Bacteriophages used to detect low levels of bacteria may be eitherchemically treated with agents such as NaOH or be selected for naturalmutations that increase their sensitivity to chemical or physicalinactivation agents. Such mutants would facilitate the inactivation ofresidual bacteriophage after primary infection of the target bacteriaand prior to amplification of those phage released from those targetbacteria.

The permissive bacteria used to amplify bacteriophage released from thetarget bacteria, may be selected for natural mutations that attenuateany pathogenic potential and/or ability to compete effectively in thenatural environment.

b) Single cycle infection and assay

Indicator microorganisms such as the enteric group of bacteria aretypically present in foods and environmental samples at levels well inexcess of specific pathogens. Increasing levels of indicator bacteriamay be utilised as a measure of an increasing probability of thepresence of pathogens and, in consequence, careful monitoring can proveof considerable value in establishing hygiene status and in HACCPmonitoring (see Microorganisms in Foods 4; ICMSF). Monitoring thepresence of indicator bacteria at levels of 10² /g or cm² would best beachieved by the phage amplification assay described above. Levels ≧10³/g or cm², however, could be assayed using a single cycle of phageinfection without amplification and, in consequence, in a time scale ofless than 100 min.

Samples containing indicator bacteria ≧10³ /g or cm² would be treatedwith an indicator associated bacteriophage(s) at a concentrationsufficient to ensure rapid infection of the indicator (m.o.i. of 10 orgreater). After 10 to 15 min to allow infection of phage DNA into theindicator bacteria, a chemical or physical treatment would be used todestroy all remaining bacteriophage. After neutralisation of thevirucidal treatment, incubation would be continued in microbial growthmedium for 40-50 min to allow completion of the phage infection cycle.Bacteriophage would be released at levels of 10⁴ or greater (assuming aminimum burst size of 10). At these levels the presence of bacteriophagecould be assayed for directly by the novel assay format described abovewithout further amplification through a permissive host.

c) Competitive binding and assay

An alternative format to that described in single cycle infection andassay, could employ competitive binding of bacteriophage between theindicator bacteria present in the sample under assay and the bacteriaengineered to detect the presence of phage. Given an equal concentrationof natural indicator bacteria and engineered assay bacteria andsub-saturating level of bacteriophage, the phage would be equallydistributed between binding and infection of indicator and assaybacteria. Under these circumstances the amount of bioluminescenceobtained from a luxAB assay would be half that obtained if there were noindicator bacteria present. In this format the assay time would be lessthan 60 min, there would be no requirement for a virucidal treatmentstep and, by manipulating the assay bacteria and bacteriophage ratio, aquantitative estimate of bacterial numbers could be established.

2. Rapid detection of environmental bacteriophage

The bacteriophage assay format could be designed so that the reporterbacteria were engineered to be responsive to infection by previouslyspecified environmental bacteriophage. Such phage might includecoliphage. Such an assay would allow the rapid detection of viablecoliphage in water and sewage effluent. Since the assay can in principlebe designed to detect any bacterium/bacteriophage couple, the detection,in situ, of any relevant bacteriophage can be contemplated.

3. Rapid evaluation of virucidal agents

The assay format of bacteriophage detection, detects only viable andinfective phage, it reflects biological activity, not merely thepresence of phage particles. The determination and assessment ofantiviral activity in potential virucides generally require elaboratecell culture or electron microscopy facilities. Bacteriophage, asprokaryotic viruses, may be used as model virus agents to test thepotency of virucidal compounds. Virucidal activity against wild typebacteriophages would be measurable as a decrease in bioluminescencefollowing assay with a bacterium genetically engineered to contain apromoter/luxAB chimera dependent for expression on phage infection. Sucha bacterium has been described herein as a novel assay format forbacteriophage.

Previously, a virucide assay using recombinant bacteriophage engineeredto contain luxAB within the bacteriophage genome has been described(Jassim et al., 1990). The present invention, however, allows the use ofwild type bacteriophage while retaining the expression ofbioluminescence as a measure of virus viability.

The present invention is further illustrated by the following Examples:

Example 1 describes the preparation and use of reporter bacteria basedon Listeria monocytogenes 23074.

Examples 2 to 5 describe techniques for killing extracellularbacteriophage, without affecting the ability of phage-infected E. colito be subsequently amplified.

Example 6 describes the detection of Listeria by activation of reporterbacteria from Example 1 with amplified bacteriophage.

EXAMPLE 1 Promoter Cloning from the B1 Phage of Listeria Monocytogenes23074

Isolation of Plasmid DNA and Phage DNA

Plasmid DNA was isolated by standard procedures and purified bycentrifugation to equilibrium in CsCl gradients. B1 phage particles werepropagated in Listeria monocytogenes ATCC 23074 and purified by bandingin CsCl gradients according to Audurier et al., (1977). DNA wasliberated from the phage by a phenol/chloroform extraction procedure andrecovered by precipitation with ethanol in the presence of 0.3M sodiumacetate.

Preparation of a B1 phage library in pSB292 for promoter screening

DNA from the B1 phage was digested separately with the restrictionenzymes AluI, HaeIII, and RsaI. After the reactions had gone tocompletion the enzyme was removed by phenol/chloroform extraction andthe DNA recovered by ethanol precipitation. In parallel, the plasmidpSB292 (FIG. 1 and Park et al. 1991) was digested with SmaI and the DNArecovered as above. A mixture of B1 phage DNA fragments was prepared bymixing equal volumes of the AluI, HaeIII and RsaI digests. Thesefragments were then ligated (insert/vector ratio 2:1) into the SmaI siteof pSB292 using a standard ligation reaction containing 1 mMhexaminecobalt III chloride. The ligation mix was dialysed againstdistilled water for 30 minutes using VSWP filters (Millipore) and usedto transform L. monocytogenes ATCC 23074 by the method of Park andStewart (1990). Transformants were recovered by plating onto brain heartinfusion agar (BHI, Oxoid) containing 5 μg/ml chloramphenicol andincubating overnight at 30° C.

Screening transformants for phage inducible promoters

Approximately 3×10³ transformants were obtained from the B1 phagelibrary. Transformants containing derivatives of pSB292 in which phagepromoters constitutively expressed luxAB were visualised, after theaddition of 20 μl dodecanol to the petri dish lid, as bioluminescentcolonies using an Argus 100 VIM3 photon imaging camera (HamamatsuPhotonics). A number of "dark" transformants (350), in which the luxgenes were not being expressed, were picked onto duplicate sets ofBHI/chloramphenicol plates and incubated at 30° C. for 4 hrs. B1 phage(10μl containing 3×10⁸ PFU) was then spotted onto one set of theduplicate plates and incubation continued for a further hour. The phageinfected and uninfected plates were then visualised under the photonimaging camera. One transformant, which was bioluminescent only in thepresence of a B1 bacteriophage infection, was considered to contain apSB292 derivative in which a phage inducible promoter was directingexpression of the lux genes. This plasmid was designated pSP19.

Promoter induction experiments

Cells containing pSP19 were grown in BHI broth containing 5 μg/mlchloramphenicol at 30° C. with shaking. When the Absorbance at 600 nmwas 0.1, cells were harvested (8000 g and 30° C. for 10 min) Thesupernatant was discarded and the cell pellet resuspended in 1/100 ofthe original volume of broth. Two 0.5 ml aliquots of this suspensionwere removed to plastic test tubes. To one tube B1 bacteriophage wasadded at a multiplicity of infection of 3. Both tubes were thenincubated at 30° C. for 10 min with no agitation to allow phageabsorption. After this period the contents of the test tubes weretransferred to separate flasks containing 50 ml of pre-warmed BHI. Thecultures were then incubated under the original conditions (30° C., 150rpm). At timed intervals, 1 ml samples were removed for cell density andcellular bioluminescence measurements. Bioluminescence was assessed byadding 0.01 vol of a 1% dodecanal solution in ethanol to samples andimmediately assessing the light production in a luminometer (TurnerDesigns, 20).

Listeria monocytogenes 23074 containing the plasmid pSP19 was infectedwith bacteriophage 23074B1 at time zero. Increasing bioluminescence wasobserved with time after phage infection. The results are shown in FIG.2.

EXAMPLE 2

Method

Phage Lambda was exposed to linoelaidic acid (LA) (50 μg/ml) in Tris-Cubuffer (1.21 g tris; 5.8 g NaCl; 0.075 g CaCl₂ ; 10 ml of 1% w/v CuSO₄per liter; pH 7.4) under differing illumination conditions. Theseincluded dark, fluorescence lighting and exposure to relatively specificwavelengths by using a range of filters with transmission optima at 300,338, 360, 375, 395, 400, 420, 450, 455 and 530 nm (Waters Associates,Inc. Manual No. 1M82902, 1980 and FIG. 3). The incident light for filtertransmission was obtained from a microscope condenser. Time of exposure,temperature of exposure and the importance of Cu²⁺ were assessed.

Results

At a concentration of 50 μg/ml, LA inactivated 93% of Lambda phage in 30minutes at 37° C. (Table 1). This inactivation was not observed ifsamples were incubated in the dark. Significantly, either with orwithout illumination, E. coli W3110 was not affected by the unsaturatedfatty acid.

                                      TABLE 1                                     __________________________________________________________________________    Effect of the combination of 50 μg/ml linoelaidic acid (LA) and light      on the                                                                        kinetic killing of phage Lambda and host cells at 37° C. for 30        minutes.                                                                                               In the presence of                                           In the absence of light                                                                        fluorescent light                                            Tris-Cu buffer                                                                        Tris-Cu buffer                                                                         Tris-Cu buffer                                                                        Tris-Cu buffer                               Microorganisms                                                                        deprived LA                                                                           supplemented LA                                                                        deprived LA                                                                           supplemented LA                              __________________________________________________________________________    Phage Lambda                                                                          3 × 10.sup.10 pfu/ml                                                            4 × 10.sup.10 pfu/ml                                                             3 × 10.sup.10 pfu/ml                                                            4 × 10.sup.10 pfu/ml                   E. coli W3110                                                                         3 × 10.sup.8 cfu/ml                                                             3 × 10.sup.8 cfu/ml                                                              4 × 10.sup.8 cfu/ml                                                             3 × 10.sup.8 cfu/ml                    __________________________________________________________________________

EXAMPLE 3

This example illustrates the use of acetic acid as a selective virucidalagent.

Into sterile Eppendorf tubes were dispensed 8.4 μl phage of 10⁹pfu/ml+8.4 μl of a particular dilution of host bacterial cells. Thismixture was incubated on ice for 10 minutes. Then to each tube was addedeither:

i) Vinegar solution to give a final concentration of vinegar of 5, 10,15 or 20% v/v in Lambda buffer, or

ii) Glacial acetic acid solution to give final concentrations of aceticacid of 0. 005, 0.05, 0.1, 0.25 and 0.5% v/v in Lambda buffer. The tubeswere further incubated at 37° C. under static conditions for 15 or 30minutes. Then the acid was neutralised by the use of 3M sodiumhydroxide. Phage growth (pfu/ml) was detected by plaque formation onplates with 100 μl of sensitive host cell (10 cfu/ml).

Results are set out in Table 2 below, and indicate that rapid (15minutes) phage killing occurs at pHs between 2.91 and 2.35, similar toresults obtained for vinegar (results not shown). Again, as for vinegar,the acetic acid system was equally effective against Listeriamonocytogenes phage.

A comparative study between 5% v/v vinegar and 0.25% v/v acetic acidshowed almost equivalent phage inactivation profiles. The preferred timefor bacterial exposure to phage was shown to be in the range of 5 to 50minutes on ice, with an optimum of about 10 minutes on ice. With thisprotocol, bacteria are detectable in 5 hours by plaque formation, asshown in the following Table 3. Even with infection periods as long as20 minutes on ice, there was shown to be no subsequent loss of bacterialviability during the 15 minutes treatment with vinegar or acetic acid.

The ability to use acetic acid mediated phage killing to detect E. coliW3110 in a mixed population of bacteria was investigated. Mixed culturescontaining 10⁹ cfu/ml of Staph.epidermis, S.arizonae, Strep.mutans,Ps.aeruginosa, and B.subtilis were inoculated with differing amounts ofE. coli W3110. Phage Lambda was employed, using the acetic acid mediatedphage inactivation protocol described above, to detect specifically theE. coli W3110 present in the mixed cultures. Table 4 documents theresults which prove that the method can be translated into practice.

                  TABLE 2                                                         ______________________________________                                        Effect of different concentrations and ph-values of acetic acid               on the survival of phage and host cells at 37° C./15 minutes.          % of glacial                                                                  acetic acid in        cfu/ml of  pfu/ml of                                    Lambda buffer                                                                            pH-values  E. coli W3110                                                                            phage Lambda                                 ______________________________________                                        0 (Lambda buffer)                                                                        7.4        3 × 10.sup.8                                                                       3 × 10.sup.9                           0.005      4.1        6 × 10.sup.8                                                                       9 × 10.sup.8                           0.05       3.24       3 × 10.sup.8                                                                       1.5 × 10.sup.7                         0.1        2.91       5 × 10.sup.8                                                                       7 × 10.sup.3                           0.25       2.57       4 × 10.sup.8                                                                       0 (0)                                                              (5.7 × 10.sup.8)                                  0.5        2.35       6 × 10.sup.7                                                                       0  .sup.                                     ______________________________________                                    

Data within brackets cfu/ml for Listeria monocytogenes ATCC 23074 andpfu/mi of phage 23074 B1 .

                  TABLE 3                                                         ______________________________________                                        Number of pfu of phage 23074 B1 obtained after the infection                  of various number of Listeria monocytogenes ATCC 23074.                       Phage      Absence of                                                         inactivation by:                                                                         host cell 100 host cells                                                                            1000 host cells                              ______________________________________                                        Lambda buffer                                                                            Complete  Complete lysis                                                                            Complete lysis                                          lysis                                                              0.25% acetic acid                                                                        No plaques                                                                              120         800                                          0.3% acetic acid                                                                         No plaques                                                                              150         750                                          15% vinegar                                                                              No plaques                                                                              110         900                                          ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        The detection of E. coli W3110                                                in complex cultures by plaque formation                                                    Approximate cfu of                                               Phage inactivation                                                                         E. coli W3110 in mixed culture                                   regimen      100       1,000  10,000                                          ______________________________________                                        0.25% acetic acid                                                                          111         700  Complete lysis                                  5% vinegar   132       1,000  Complete lysis                                  ______________________________________                                    

EXAMPLE 4

Protocol

An overnight culture of E.coli W3110 was diluted ten-fold stepwise inLambda buffer. 8.4 μl aliquots containing different numbers of cellswere transferred in duplicate to sterile Eppendorf tubes. To this, 8.4μl of a suspension of phage Lambda (1×10⁹ pfu/ml) was added and thephage allowed to adsorb to the cells for 10 minutes at 0° C.Non-adsorbed phage were then inactivated by the addition of 83.2 μl ofacetic acid in Lambda buffer (to give a final concentration of 0.25%)and incubation at 39° C. for 20 minutes. The acid was then neutralisedby the addition of 14 μl of 0.3M NaOH (to give final pH of ≈8.0).

Unaffected phage (i.e. those protected by adsorption to W3110 cells)were then immediately titred in one of the duplicate sets by mixing with100 μl of W3110 cells (1×10⁶ cfu) and 3 ml of 0.6% top layer agar andpoured over Luria agar plates and incubated at 37° C. Plaques werecounted after 18 hrs incubation. Phage amplification for the otherduplicate set, was initiated by the addition of 3 ml of FT broth andcultures incubated at 37° C. with shaking (150 rpm) for 5 hrs. Phagetitres were determined by plating 100 μl samples as previouslydescribed.

Results are set out in the following Table 5.

Lambda buffer is 6 mM Tris-HCl, pH 7.8 10 mM MgSO₄. 7H₂ O, 10 mM CaCl₂,0.005% gelatin.

FT broth is per liter 5 g NaCl, 10 g Tryptone, 10 ml 1M MgCl₂, 20 ml 10%maltose.

                  TABLE 5                                                         ______________________________________                                        Phage Lambda infection of various numbers of                                  E. coli W3110 and the amplification obtained in 5 hrs at                      37° C. subsequent to external phage killing                                     Approximate cfu of E. coli W3110                                              0   7        70       700    7000                                    ______________________________________                                        On solid medium                                                                          nil   2        7      200    1000                                  Time zero                                                                     In liquid medium                                                                         nil   4 × 10.sup.5                                                                     5 × 10.sup.5                                                                   2 × 10.sup.6                                                                   2 × 10.sup.9                    5 hrs                                                                         (phage                                                                        amplification)                                                                ______________________________________                                    

These Examples 3 and 4 describe a complete protocol wherebybacteriophage can be killed within 15 minutes, at an efficiency greaterthan 9 log cycles, without any detectable reduction in the number ofcoexistent bacterial cells or in their ability to amplify aftervirucidal treatment. The Examples further show that the method isapplicable to a variety of bacteriophage and is effective for both Grampositive and Gram negative genera. The direct detection of bacteria bybacteriophage amplification in axenic and complex cultures isdemonstrated.

Some minor variations of protocol may be necessary depending on theorganism under test but the principle remains the same, as the followingExample shows.

EXAMPLE 5

The technique of Examples 3 and 4 was repeated, but using Staphyloccusaureus NCIMB 8588 and phage NCIMB 9563. Differential phage killing wasachieved using two different concentrations of acetic acid. After phagebinding at 0° C., the samples were warmed to 37° C. for 5 minutes, andthen cooled to 0° C. prior to acetic acid treatment. Results are set outin Table 6.

                  TABLE 6                                                         ______________________________________                                        % acetic acid                                                                 used for phage                                                                            Number of S. aureus cells                                         inactivation                                                                              0        8     80     800  8000                                   ______________________________________                                                  Number of plaques obtained                                          0.15%       0        8     80     800  8000                                   0.25%       0        8     80     800  8000                                   ______________________________________                                    

EXAMPLE 6

Detection of Listeria by Activation of Lm23074 (pSP19 ) with amplifiedPhage

Protocol

An overnight culture of L. monocytogenes 23074 (ATCC) containing 6×10⁸cells per ml was diluted ten-fold stepwise in Lambda buffer. Tenmicroliter aliquots containing different number of cells weretransferred to sterile Eppendorf tubes and cooled on ice-water for 5minutes. To this, 10 μl of a suspension of phage Lm23074B1 (ATCC)containing 1×10⁹ pfu/ml was added and the phage allowed to adsorb to thecells for 10 minutes at 0° C. Non-adsorbed phage were then inactivatedby the addition of 80 μl of 0.31% acetic acid in Lambda buffer andincubation at 39° C. for 20 minutes. The acid was then neutralised bythe addition of 14 μl of 0.3M NaOH (to give a final pH of ≈8.0). Toallow amplification of phage, 1×10⁸ cells of the propagating strainLm23074 in 1.4 ml of Luria broth were added to the Eppendorf and thesamples incubated at 30° C. for 4 hours. The number of phage present inthis culture was titred initially, directly after addition of thepropagating bacteria, by spotting 10 μl samples on to a lawn of Lm23074(this method of assay only allows titres to be determined down to 10³pfu/ml). The presence of phage in the supernatant after 4 hoursamplification was detected by the addition of 100 μl aliquots to 200 μlof an exponentially growing culture of Lm23074 (pSP19 ) at an A₄₅₀ ofbetween 0.25 and 0.3. Infection was allowed to proceed for 60 minutesbefore light induction was measured.

Results are set out in the following Table 7, in which the light unitsrecorded for each cell are given in column 3.

                  TABLE 7                                                         ______________________________________                                        Initial No.  Initial   Light units after                                      of bacteria  Phage titre                                                                             4 hrs amplification                                    ______________________________________                                        6 × 10.sup.6                                                                         1.8 × 10.sup.6                                                                    20818                                                  6 × 10.sup.5                                                                         9.0 × 10.sup.5                                                                    14243                                                  6 × 10.sup.4                                                                         1.8 × 10.sup.4                                                                    4458                                                   6 × 10.sup.3                                                                         9.0 × 10.sup.2                                                                    3601                                                   6 × 10.sup.2                                                                         nd         192                                                   60           nd        1054                                                   6            nd         268                                                   0            nd         42                                                    0            nd         32                                                    ______________________________________                                         nd: not detectable in titre system used.                                 

The above example shows that as few as 6 cells of Listeria monocytogenescan be detected in a total assay time of 5.5 hrs. This is a majoradvance in current rapid methods of detection.

REFERENCES

Audurier, A., Rocourt, J. and Courtieu, L. 1977 Isolement etcaracterisation de bacteriophages de Listeria monocytogenes. Ann.Microbiol. (Inst. Pasteur), 127A, 185-198.

Chamberlin, M., McGrath, J., and Waskell, L., 1970, New RNA polymerasefrom Escherichi coli infected with bacteriophage T7, Nature 228: 227.

Gasson, M. J., and Anderson, P. H., 1985, High copy number plasmidvectors for use in lactic streptococci, FEMS Microbiol. Lett.30: 193.

Hirotani, H., Ohigashi, H., Kobayashi, M., Koshimizu, K. & Takahashi, E.1991 Inactivation of T5 phage by cis-vaccenic acid, an antivirussubstance from Rhodopseudomonas capsulata, and by unsaturated fattyacids and related alcohols. FEMS Microbiology Letterd 77, 13-18.

Jacob, F., and Monod, J., 1961, Genetic regulatory mechanisms in thesynthesis of proteins, J. Mol. Biol.t: 318.

Jassim, S. A. A., Ellison, A., Denyer, S. P., and Stewart, G. S. A. B.,1990, In vivo bioluminescence: a cellular reporter for research andindustry, J. Biolum, Chemilum. 5: 115.

McKnight, S., and Tjian, R., 1986, Transcriptional selectivity of viralgenes in mammalian cells, Cell 46: 795.

Old, R. W., and Primrose, S. B., 1989, Bacteriophage and Cosmid vectorsfor E. coli, in: Principles of Gene Manipulation 4th edn., pp. 60-86,Blackwell Scientific Publications.

Park, S. F. and Stewart G. S. A. B. 1990 High efficiency transformationof Listeria monocytogenes by electroporation of penicillin treatedcells. Gene 94 129-132.

Park, S. F., Nissen U. and Stewart G. S. A. B. 1991 The cloning andexpression of luxAB in Listeria monocytogenes in: Bioluminescence andChemiluminescence current status (Stanley, P. E. and Kricka, L. J. eds)pp 35-38 (John Wiley and Sons).

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Rabussay, D., and Geiduschek, E. P., 1977, Regulation of gene action inthe development of lyric bacteriophages, in: Comprehensive Virology 8(F. C. Wagner, ed.), pp. 1-150, Plenum Press, New York.

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We claim:
 1. The method of testing for the presence or concentration ofa target bacteria in a fluid sample by the steps of:a) adding to thesample bacteriophage capable of infecting the target bacteria underconditions to cause it to infect any target bacteria present. b)destroying, removing, neutralizing or inactivating extracellularbacteriophage in the sample, c) incubating the sample to completeinfection and cause the target bacteria to release the bacteriophage,and d) assaying the bacteriophage as an indication of the presence orconcentration of the target bacteria in the sample.
 2. The method asclaimed in claim 1, including between steps c) and d) an additionalstepci) amplifying, by the use of a permissive bacterial host, thebacteriophage in the sample.
 3. The method as claimed in claim 2,wherein the target bacterium is a pathogen.
 4. The method as claimed inclaim 1 to wherein step b) is performed by subjecting the sample tophoton irradiation in the presence of an unsaturated fatty acid.
 5. Themethod as claimed in claim 1 to wherein step b) is performed by means ofacetic acid.
 6. The method as claimed in claim 5, wherein step a) isperformed at a temperature below ambient.
 7. The method as claimed inclaim 1, wherein the resulting bacteriophage is capable of infectingListeria monocytogenes and step d) is performed bydi) using theresulting bacteriophage to infect reporter bacteria which are Listeriamonocytogenes genetically engineered to have an indicator gene which isa promotorless lux gene under the expression control of a temporallyregulated bacteriophage gene switch which on expression gives rise toluciferase, wherein expression of the indicator gene is initiated bybacteriophage infection of the bacteria and dii) observing thedetectable polypeptide expressed by the reporter bacteria as anindication of the presence or concentration of the target bacteria inthe sample.